Project Summary The overarching goal of this project is to detect recurrent bladder cancer using a unique technology platform capable of quantitating tumor-specific biomarkers in patient urine. If successful, this testing platform will change the current paradigm for monitoring recurrent bladder cancer. Following treatment for bladder cancer, patients face up to a 60% recurrence rate, and are typically monitored every few months by cytoscopy and single biomarker measurements using expensive central laboratory testing. Unfortunately, cytoscopy is invasive, and has a complication rate as high as 15%. Patient compliance is therefore a significant issue. Our goal is to translate a new technology from the PI and Co-I's laboratories into the clinic where it will offer rapid, near patient, sensitive, specific, non-invasive and inexpensive testing for the detection of recurrent bladder cancer. The Weiss and Penner laboratories have recently described Electro-Phage biosensors that use customized viruses (bacteriophage or phage) as biomarker affinity reagents. The biosensor can bind and measure the concentrations of cancer biomarkers in synthetic urine and spiked urine samples from anonymous donors. We have combined viruses with an electrically conductive polymer, which allows impedance spectroscopic measurements for the robust detection and quantification of sub-nM concentrations of a prostate cancer biomarker in urine. The pM assay sensitivity is within the requirements for clinical testing. This sensitivity is achieved by coating the phage with both genetically encoded and chemically synthesized binders to the putative cancer biomarkers. The proposed project expands on previous studies to include multiple cancer biomarkers and identify a molecular ?fingerprint? for recurrent bladder cancer. We hypothesize that a multi- analyte approach will allow better sensitivity and specificity for monitoring recurrence of bladder cancer. The targeted cancer biomarkers will be over-expressed, purified, and refolded; then phage display will be employed to identify tumor specific binders. In parallel, experiments will optimize the architecture of the Phage-Electrode to maximize signal-to-noise, and improve the sensitivity and specificity of the multi-analyte assay. Such optimization will be used to guide development of a multi-channel sensor, fabricated by the industrial partner PhageTech. The clinical trial will be conducted in two stages, and focus on analytical and clinical validation of the Electro-Phage biosensor for the detection of bladder cancer recurrence. In Stage 1, urine samples from 10-20 patients will be analyzed to compare sensitivity and quantification with FDA-approved tests for two biomarkers; in addition, Stage 1 will collect sufficient data to guide the design of the Stage 2 clinical trial. In this larger, pilot clinical trial, approximately 200 patients with bladder cancer of all pathologic stages and histologic grades will be enrolled; an equal number of control samples will be obtained from patients with other genito-urinary tract malignancies and healthy patients. Thus, the study will compare current monitoring modalities for recurrent bladder cancer with the novel biosensor array approach, a distinct, urine-based, molecular fingerprint diagnostic for bladder cancer recurrence. This point of care, easy to perform, label- and reagent-free sensing approach will allow for non-invasive, less costly, more frequent, monitoring and therefore earlier detection of recurrent bladder cancer.